A microtubule-dependent zone of active RhoA during cleavage plane specification.

Bement WM, Benink HA, von Dassow G - J. Cell Biol. (2005)

Bottom Line:
Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis.Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement.We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.

ABSTRACTCytokinesis in animal cells results from the assembly and constriction of a circumferential array of actin filaments and myosin-2. Microtubules of the mitotic apparatus determine the position at which the cytokinetic actomyosin array forms, but the molecular mechanisms by which they do so remain unknown. The small GTPase RhoA has previously been implicated in cytokinesis. Using four-dimensional microscopy and a probe for active RhoA, we show that active RhoA concentrates in a precisely bounded zone before cytokinesis and is independent of actin assembly. Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis. Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement. We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.

fig4: RhoA activation in the equator does not depend on actin assembly. All times in this figure are in minutes:seconds after the addition of cytochalasin D. (A) Projection of 18 1-μm sections through a 16-cell purple urchin embryo treated with 10 μM cytochalasin D during prometaphase. As the drug takes effect, cell surfaces become spiked and ruffled. Cells fail to develop a furrow but, nevertheless, form zones of RhoA activation (arrowheads, 28:40). Bright, glowing objects in other blastomeres are nuclei that accumulate eGFP probes nonspecifically (see Fig. 2, C and D). (B) Projection of four sections through a purple urchin embryo to which 10 μM cytochalasin D was added shortly before furrowing. Furrows with associated RhoA activity develop (arrowheads) and regress, but equatorial RhoA activity remains high despite furrow regression. (C) Projection of 20 sections through the near surface of the embryo shown in B; although the cortex of cytochalasin D–treated cells is wildly deranged, RhoA remains elevated in the equator (arrowheads). (D) Projection of 15 sections through a green urchin embryo to which 10 μM cytochalasin D was added at metaphase. Zones of RhoA activity (arrowheads) appear on schedule despite the absence of ingression. (E) A more extreme case than D; projection of 10 1-μm sections through one blastomere of an eight-cell green urchin embryo attempting to cleave in 12-μM cytochalasin D. Active RhoA appears on tubular extensions projecting inward from the cortex, most prominently near the equator. (F) Projection of 38 sections through an eight-cell green urchin embryo treated identically to that in E. Tubular projections point toward the spindle poles (asterisks). See online supplemental material for videos corresponding to A (Video 6) and E (Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Bars, 25 μm.

Mentions:
If the RhoA activity zone represents the link between microtubules and cytokinetic apparatus specification rather than a consequence of equatorial actin accumulation, the zone should form even under conditions in which actin cannot accumulate. We treated urchin embryos with cytochalasin D to prevent actin assembly. In purple urchin embryos, a 15–20-min treatment with cytochalasin dramatically reduced the F-actin level, and furrows failed to develop (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Although the cortex of these cells became obviously disrupted, forming dramatic ruffles and pleats as mitosis progressed, a RhoA zone nevertheless formed at the equator (Fig. 4 A and Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Latrunculin B likewise prevented furrowing and deranged the cortex, yet did not interfere with the formation of the equatorial RhoA zone (Fig. S3). When cytochalasin treatment begins shortly before cleavage, furrows may form and then regress. RhoA zones were maintained in regressing furrows (Fig. 4 B). Indeed, regressed zones persisted for 30 min or more in the presence of cytochalasin, which is more than three times as long as in control cells (Fig. 1), even when the cortex is obviously disrupted (Fig. 4 C). RhoA activity zones also formed in green urchin embryos that were treated with cytochalasin (Fig. 4 D). These results show that the equatorial activation of RhoA causally precedes F-actin accumulation.

fig4: RhoA activation in the equator does not depend on actin assembly. All times in this figure are in minutes:seconds after the addition of cytochalasin D. (A) Projection of 18 1-μm sections through a 16-cell purple urchin embryo treated with 10 μM cytochalasin D during prometaphase. As the drug takes effect, cell surfaces become spiked and ruffled. Cells fail to develop a furrow but, nevertheless, form zones of RhoA activation (arrowheads, 28:40). Bright, glowing objects in other blastomeres are nuclei that accumulate eGFP probes nonspecifically (see Fig. 2, C and D). (B) Projection of four sections through a purple urchin embryo to which 10 μM cytochalasin D was added shortly before furrowing. Furrows with associated RhoA activity develop (arrowheads) and regress, but equatorial RhoA activity remains high despite furrow regression. (C) Projection of 20 sections through the near surface of the embryo shown in B; although the cortex of cytochalasin D–treated cells is wildly deranged, RhoA remains elevated in the equator (arrowheads). (D) Projection of 15 sections through a green urchin embryo to which 10 μM cytochalasin D was added at metaphase. Zones of RhoA activity (arrowheads) appear on schedule despite the absence of ingression. (E) A more extreme case than D; projection of 10 1-μm sections through one blastomere of an eight-cell green urchin embryo attempting to cleave in 12-μM cytochalasin D. Active RhoA appears on tubular extensions projecting inward from the cortex, most prominently near the equator. (F) Projection of 38 sections through an eight-cell green urchin embryo treated identically to that in E. Tubular projections point toward the spindle poles (asterisks). See online supplemental material for videos corresponding to A (Video 6) and E (Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Bars, 25 μm.

Mentions:
If the RhoA activity zone represents the link between microtubules and cytokinetic apparatus specification rather than a consequence of equatorial actin accumulation, the zone should form even under conditions in which actin cannot accumulate. We treated urchin embryos with cytochalasin D to prevent actin assembly. In purple urchin embryos, a 15–20-min treatment with cytochalasin dramatically reduced the F-actin level, and furrows failed to develop (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Although the cortex of these cells became obviously disrupted, forming dramatic ruffles and pleats as mitosis progressed, a RhoA zone nevertheless formed at the equator (Fig. 4 A and Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Latrunculin B likewise prevented furrowing and deranged the cortex, yet did not interfere with the formation of the equatorial RhoA zone (Fig. S3). When cytochalasin treatment begins shortly before cleavage, furrows may form and then regress. RhoA zones were maintained in regressing furrows (Fig. 4 B). Indeed, regressed zones persisted for 30 min or more in the presence of cytochalasin, which is more than three times as long as in control cells (Fig. 1), even when the cortex is obviously disrupted (Fig. 4 C). RhoA activity zones also formed in green urchin embryos that were treated with cytochalasin (Fig. 4 D). These results show that the equatorial activation of RhoA causally precedes F-actin accumulation.

Bottom Line:
Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis.Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement.We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.

ABSTRACTCytokinesis in animal cells results from the assembly and constriction of a circumferential array of actin filaments and myosin-2. Microtubules of the mitotic apparatus determine the position at which the cytokinetic actomyosin array forms, but the molecular mechanisms by which they do so remain unknown. The small GTPase RhoA has previously been implicated in cytokinesis. Using four-dimensional microscopy and a probe for active RhoA, we show that active RhoA concentrates in a precisely bounded zone before cytokinesis and is independent of actin assembly. Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis. Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement. We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.